methods
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Methods

     

Our work involves the use of several complementary methods:

     

3D Anatomy

The neurogenic subventricular zone is a complex 3-dimentional structure. Moreover, it appears to be highly heterogeneous, with stem cells located in different microdomains of the SVZ giving rise to distinct cell types, both glial and neuronal.
Studying cellular specification in the subventricular zone therefore requires the establishment of new methods that allow identifying and quantifying neural stem cell subtypes and their progeny.

We are approaching this problematic by combining immunodection of defined transcription factors with serial sectioning and 3 dimensional reconstructions of the forebrain ventricles. Chosen transcription factors define distinct, non-overlapping populations of neural stem/progenitor cells, or cells of the same lineage, but at different stages of their differentiation. Their superimposition to the ventricles outline reveals their distribution in the SVZ in the naïve animal or after experimental manipulation.

 

Molecular biology

Neural stem cells specify in distinct cellular cell types because of the coordinated expression of transcription factors.
In the last decade, sequencing of several mammalian genomes combined with advances in high throughput techniques has represented a major breakthrough in molecular biology. DNA microarrays allow the identification of new genes, based on their expression levels in defined cell types.

We are combining microdissections with fluorescent or magnetic cell sorting to isolate populations of cells with different degrees of enrichment. Comparison of the transcriptome of these cell populations allows the identification of genes likely to be involved in key steps of the cellular differentiation process, including the specification of different cell types.

 

Have a look at the movie below showing one of our 3D reconstructions of the brain ventricules.

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Gene manipulation

Gene manipulation relies on the intracellular delivery of DNA. The method of choice to label neural stem cells in the adult central nervous system is the stereotoxic delivery of retroviral viruses. This technique is however time and resources consuming. Over the last years, electroporation of the lateral ventricle has become our method of choice to deliver genetic constructs to neural stem cells of the lateral ventricle. This approach offers the flexibility of concomitantly manipulating the expression of single or multiple genes.

We have recently refined and improved the electroporation technique by showing that it can be accurately and reproducibly used to target transgene delivery to specific walls of the LV. Fate mapping the progeny of radial glial cells (RG, the perinatal neural stem cells) located in distinct walls of the postnatal LV provides a baseline onto which future studies aiming at investigating the role of factors in neuronal specification can be compared.

 

Imaging

We are making use of a number of transgenic animals to visualize and fate map defined population of cells in the developing as well as postnatal central nervous system. Among them are the Mash1::GFP mice that express fluorescent protein in type C, i.e. fast proliferating progenitors, as well as their early progeny. Other mice are the Neurog2kiGFP mice. We have used this mouse strain to unravel the pattern and timing of expression of Neurog2 in granule cell progenitor in the adult hippocampus. More recently, this mouse strain has allowed us to identify a new population of glutamatergic progenitors in the adult subventricular zone.

 

 

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Beside straight reporter mice, we are also making increasing use of Cre-lox mice that allow the conditional but permanent expression of a reporter gene in selected progenitor lineages. For example, Neurog2CreERT2 animal, into which Cre expression and activity is controlled by Neurog2 genomic sequences but also by the injection of Tamoxifen to the animals, allowing one to fate map progenitors expressing Neurog2 at different time of the development.
Most of our imaging is done on fixed tissue, using epifluorescence microscopy combined with deconvolution processing of the images, as well as confocal microscopy.

We are also performing live imaging of dendritic spines, by using confocal microscopy in long term organotypic slice cultures.